Atomic models by cryo-EM and site-directed spin labeling: application to the N-terminal region of Hsp16.5 |
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Authors: | Koteiche Hanane A Chiu Steve Majdoch Rebecca L Stewart Phoebe L Mchaourab Hassane S |
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Institution: | Department of Molecular Physiology and Biophysics, Center for Structural Biology, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA. |
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Abstract: | We report an approach for determining the structure of macromolecular assemblies by the combined application of cryo-electron microscopy (cryo-EM) and site-directed spin labeling electron paramagnetic resonance spectroscopy (EPR). This approach is illustrated for Hsp16.5, a small heat shock protein that prevents the aggregation of nonnative proteins. The structure of Hsp16.5 has been previously studied by both cryo-EM and X-ray crystallography. The crystal structure revealed a roughly spherical protein shell with dodecameric symmetry; however, residues 1-32 were found to be disordered. The cryo-EM reconstruction at 13 A resolution appeared similar to the crystal structure but with additional internal density corresponding to the N-terminal regions of the 24 subunits. In this study, a systematic application of site-directed spin labeling and EPR spectroscopy was carried out. By combining the EPR constraints from spin label accessibilities and proximities with the cryo-EM density, we obtained an atomic model for a portion of the Hsp16.5 N-terminal region in the context of the oligomeric complex. |
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